Characterization of a New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine

In this study, a gene encoding β-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The Vmax, Km, kcat, and kcat/Km of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 μmol min–1 l–1, 39.99 μmol l–1, 4667.07 s–1, and 116.71 ml μmol–1 s–1, respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)2–(GlcNAc)6) into GlcNAc with hydrolysis ability order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6. Moreover, NAGaseA could generate (GlcNAc)3–(GlcNAc)6 from (GlcNAc)2–(GlcNAc)5, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.

INTRODUCTION
Chitin is the second most abundant polysaccharide on earth after cellulose, it is mainly derived from fungal cell walls, insect exoskeletons, and the crab and shrimp shells. An estimated 10 10 -10 11 tons of chitin are produced per year (Anitha et al., 2014). However, 35-45% of chitin biomass is discarded as waste due to a lack of efficient refinery protocols, which leads to waste of resources and severe environmental problems (Wei et al., 2017;Zhou et al., 2017a). N-acetyl glucosamine (GlcNAc), the monomeric unit of chitin, possesses many specific bioactivities and has been widely used in biomedical, food, and chemical industries (Bhattacharya et al., 2007;Suresh and Kumar, 2012;Kisiel and Kepczynska, 2017). Therefore, it is of economic and environmental value to realize the efficient production of GlcNAc from abundant chitin resources (Gao et al., 2018).
Commercial GlcNAc was often produced via acid hydrolysis of chitin. However, this protocol is difficult to directly obtain GlcNAc owing to the deacetylation of the N-acetyl group of products (Aam et al., 2010). In this case, chitin is first hydrolyzed to GlcN, and then chemical acetylated to form GlcNAc. This multistep process not only results in low yield, high cost, and poor biological activity of products but also leads to numerous environmental issues (Chen et al., 2010;Kim et al., 2017). Alternatively, enzymatic hydrolysis of chitin into GlcNAc using chitinolytic enzymes was shown to be a more attractive approach in recent years, because of the green process and the excellent bioactivity of the product (Park et al., 2011).
Chitinolytic enzymes are complex enzyme systems with a good synergistic effect, which could be classified into three types: endo-acting chitinases that cut randomly chitin chains to generate N-acetyl chitin oligosaccharides (N-acetyl COSs); progressive exo-acting chitinases that release GlcNAc dimer from the non-reducing or reducing end of chitin chains; NAGases that hydrolyze N-acetyl COSs or GlcNAc dimer into GlcNAc (Zhou et al., 2017a;Lv et al., 2019;Liu et al., 2020). Among, NAGase plays a key role in the control of the ratio and yield of GlcNAc during the hydrolysis process of chitin. Thus, it is of great significance to excavate NAGase with high activity for efficiently converting N-acetyl COSs into GlcNAc.
In our previous study, chitinolytic enzymes were derived from the bacterium Chitinibacter sp. GC72 isolated from pond mud in Nanjing were capable of hydrolyzing chitin into GlcNAc as the sole product (Gao et al., 2015). Moreover, only one gene encoding NAGase, named NAGaseA, was found in strain GC72 via complete genome sequencing and analysis (Zhang et al., 2020a). In this study, the NAGaseA gene was cloned from the genome of strain GC72 and heterologously expressed in Escherichia coli (BL21). The enzymatic properties and hydrolysis mode of the recombinant NAGaseA were investigated. Furthermore, the synergetic effect between NAGaseA and various chitinases in converting chitin to produce GlcNAc was also studied. This study provided a possible application in the enzymatic production of GlcNAc.

Chemicals, Strains, and Plasmids
Chitin powder, 4-methylumbelliferyl N-acetyl glucosaminide (4-MU-GlcNAc), and pNP-acetyl galactosaminide (pNP-GlcNAc) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). N-acetyl chitooligosaccharides (N-acetyl-COSs) standards ranging from dimer to hexamer were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). The molecular reagents were purchased from Takara Bio Inc. (Dalian, China). All chemicals used in this study were of analytical grade or higher purity. Colloidal chitin was prepared from chitin powder according to the methods described by Gao et al. (2015).

Molecular Cloning and Sequence Analysis
The genomic DNA of strain GC72 was extracted using a bacteria genome extraction Kit (TIANGEN, China) and was used as the PCR template. Two primers used to amplify the NAGaseA were synthesized by Genscript Biotech (Nanjing, China) and the sequences were as followed: forward primer 5 -GTGCCGCGCGGCAGCCATATGAACAAGCCAGCAGGT-3 ; reserve primer 5 -GTGGTGGTGGTGCTCGAGCACCGCAAC CACCCGGCT-3 . The PCR system and conditions were as follows: 94 • C for 5 min, followed by 30 cycles of 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 1 min, and a final extension at 72 • C for 10 min. The NAGaseA gene generated from PCR and the plasmid pET-28a (+) was double digested with NdeI and XhoI, followed by a ligation using the ClonExpressTM II/One Step Cloning Kit (Vazyme, China). The recombinant plasmid was transformed into E. coli Trans-T1 competent cells and sequenced by Genscript Biotech (Nanjing, China).
Nucleotide and amino acid sequences were analyzed using Snap Gene TM 1.1.3 software 1 and the ExPASy protparam tool. 2 The DNA and protein sequence alignments were performed via the NCBI server with the programs BLASTN and BLASTP, 3 respectively. Phylogenetic trees were inferred using the neighborjoining algorithm in MEGA 7. The conserved domains and the GH family classification were identified via the website. 4 The signal peptide was predicted in the SignalP 4.1 server. 5 Protein homologous sequences alignment was carried out using ClustalX 2.1 software and ESPript 3.0. 6 The structure of NAGaseA was predicted with RaptorX. 7

Expression and Purification of Recombinant NAGaesA
The recombinant plasmid pET-28a (+) harboring NAGaesA gene was transformed into E. coli BL21(DE3), incubated in LB liquid medium (containing 50 µg/ml kanamycin), and then cultured at 37 • C with shaking at 200 rpm. When the optical density (OD 600 ) of the culture medium was approximately 0.6, isopropyl β-Dthiogalactoside was added to a final concentration of 1 mM for protein induction, and the culture was incubated overnight at 18 • C with shaking at 200 rpm.
Cultures were harvested by centrifugation at 6,000 × g and 4 • C for 10 min, after which the pellet was gently resuspended in binding buffer A (50 mM phosphate buffer, 500 mM NaCl, 50 mM imidazole, pH 7.4) and lysed by JY92-IIN ultrasonication (Ningbo Xinzhi Biotechnology, Ltd., Ningbo, China). The cell debris was removed by centrifugation at 12,000 × g for 10 min at 4 • C and the supernatant was retained as a crude enzyme. The recombinant NAGaseA was purified using a fast protein liquid chromatography 448 system (GE AKTA Pure 150; General Electric Co., IA, America with a Ni-449 nitrilotriacetic acid affinity chromatography (Ni-NTA) column (His Trap TM FF 5 ml). The supernatant was filtered with a 0.22 µm membrane before being loaded onto a Ni Sepharose column. The NAGaseA protein was eluted with buffer B (50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, and pH 7.4) under a flow rate of 3 ml/min. The eluted protein was collected, concentrated, and exchanged with 20 mM phosphate buffer (pH 7.0) via ultrafiltration and stored at 4 • C before use (Zhang et al., 2020b).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to identify the target protein, and the protein concentration was determined using the Bradford method (Bradford, 1976).

Enzyme Assay and Substrate Specificity of Recombinant NAGaesA
NAGaseA activity was assayed using pNP-GlcNAc as substrate. A total of 1 ml reaction mixture containing 50 µl pNP-GlcNAc (10 mM) and 10 µl of purified NAGaseA in 50 mM phosphatebuffered saline (PBS) buffer (pH 7.0). The mixture was incubated at 40 • C for 10 min and then 1 ml NaOH (1 M) solution was added to terminate the reaction. The amount of pNP released was determined under the absorbance measured at 405 nm according to our previous reported (Zhang et al., 2018). One unit of NAGase activity was defined as the amount of enzyme required to release 1 µmol pNP per minute under the assay conditions. Chitinase activity was measured using colloid chitin as the substrate. The 1 ml reaction mixture was performed with 0.2 ml of enzyme and 0.3 ml colloid chitin (10 g/l) in 50 mM phosphate buffer (pH 7.0). The reaction was conducted at 37 • C for 30 min, and then 1 ml of 3,5-dinitrosalicylic acid (DNS) was added to the mixture followed by boiling at 100 • C for 5 min (Breuil and Saddler, 1985).

Enzymatic Characterization of Recombinant NAGaseA
The enzymatic characterization of recombinant NAGaseA was performed using pNP-GlcNAc as the substrate. To determine the optimum temperature of NAGaseA, the reaction was incubated under interval temperatures ranging from 30 • C to 80 • C in 50 mM PBS (pH 6.5). The thermostability of NAGaseA was determined by measuring the residual activity at pH 6.5 and 40 • C after the enzyme was treated in 50 mM sodium citrate (pH 6.5) for 12 h at different temperatures.
The kinetics parameters were determined by measuring the enzyme activity toward pNP-GlcNAc at 40 • C in 50 mM PBS (pH 6.5) for 10 min using different concentrations of substrate (50-2,500 µM) as the substrate. The values of V max , K m , and k cat were estimated by linear regression from double-reciprocal plots according to the method of Lineweaver (Price, 1985).

Hydrolytic Pattern of Recombinant NAGaseA
The reaction mixtures containing purified NAGaseA (60 ng) and various substrates ((GlcNAc) 2 -(GlcNAc) 5 ) at a final concentration of 10 g/l were incubated at 40 • C at various time intervals. In each case, the supernatant after hydrolysis was diluted with 50% acetonitrile and centrifuged at 8,000 × g for 10 min to remove the protein. The hydrolysis products were analyzed by Agilent 1260 series HPLC system according to our previous study (Zhang et al., 2018).

Cooperative Interaction Analysis of NAGaseA With Other Chitinases
The fermentation broth of strain GC72 and SYBC-H1 was centrifuged at 12,000 × g for 15 min at 4 • C, and the supernatant was collected as a crude enzyme before use. Exochitinase ChiA from Serratia proteamaculans (stored in our laboratory) was cloned, expressed, and purified as previously reported (Purushotham et al., 2012).
The cooperative interaction between NAGaseA and other sources of chitinases derived from strain Serratia proteamaculans (recombinant ChiA), strain SYBC (crude enzyme), and strain GC72 (crude enzyme) were determined using colloid chitin as the substrate. The reaction mixture (1 ml) contained colloidal chitin with a final concentration of 10 g/l and either 50 µl NAGaseA (4.8 U/ml reaction system), 50 µl ChiA (6.1 U/ml reaction system), 50 µl SYBC chitinase (2.8 U/ml reaction system), and 50 µl GC72 chitinase (5.2 U/ml reaction system) or both enzymes and was incubated at 40 • C in 50 mM PBS (pH 6.5) for 30 min. The amount of reducing sugars released was measured using the DNS method and HPLC mentioned above.

Cloning of the NAGaseA Gene and Sequence Analysis
Based on the gene function prediction of the complete genome of Chitinbacter sp. GC72, ORF 159 was annotated as a potential β-N-acetylglucosaminidase (NAGaseA) gene. The total length of NAGaseA gene is 2, 535 bp, encoding 844 amino acids. After PCR, the same nucleic acid sequence was obtained, which indicated that NAGaseA gene was successfully cloned. Besides, the predicted molecular weight and theoretical pI of NAGaseA were 92.4 kDa and 5.24, respectively.
The result of multiple alignments of NAGaseA with other GH20 NAGases was shown in Supplementary Figure 2. The typical acidic pairs D512-E513 in NAGaseA are completely aligned with many other functionally characterized GH20 To determine the temperature stability, the enzyme was incubated in 50 mM sodium citrate (pH 6.5) for 12 h at different temperatures, the residual activity was measured at pH 6.5 and 40 • C. (C) The optimal pH of the recombinant NAGaseA. The optimal pH was determined in 50 mM solutions of various buffers within the pH range 3-11. (G Citrate buffer (pH 3.0-6.0), NPhosphate buffer (pH (6.0-8.0), L Tris-HCl buffer (pH 7.0-9.0), I Glycine-NaOH buffer (pH 8.5-10.5)). (D) The pH stability of the recombinant NAGaseA. To determine the pH stability, the enzyme was incubated with various pH buffers, and the residual activities were measured (G Citrate buffer (pH 6.0), NPhosphate buffer (pH 6.5), L Phosphate buffer (pH 7.0), I Phosphate buffer (pH 8),NGlycine-NaOH buffer (pH 9), GGlycine-NaOH buffer (pH 10).

Expression of NAGaseA Gene and Purification of Recombinant NAGaseA
The gene encoding NAGaseA was successfully expressed as a soluble protein in E. coli BL21 (DE3). The SDS-PAGE analysis (Figure 2) showed that a single target protein band was obtained with a molecular weight of ∼92 kDa after Ni-NTA resin affinity purification, which was consistent with the 92,379 Da calculated from the amino acid sequence containing the His6-tag. This is different from that of some GH20 NAGases from Microbacterium sp. HJ5 (55.9 kDa; Zhou et al., 2017b), Paenibacillus sp. (57.5 kDa), V. harveyi 650 (73 kDa; Suginta et al., 2010), and Streptomyces thermoviolaceus (60 kDa; Kubota et al., 2004). However, the M w of NAGaseA is similar to the previously reported GH20 NAGase from C. meiyuanensis with a molecular mass of 92,571 Da (Zhang et al., 2020b). The specific activity of recombinant NAGaseA exhibited a 1.39-fold increase from 270.17 to 373.29 U/mg with a protein recovery of 78.6% yield after purification (Supplementary Table 1).

Effects of Metal Ions on Activity of Recombinant NAGaseA
Many reports have shown that metal ions affected enzymatic activity. Thus, the effects of various metal ions on NAGaseA activity were also investigated. As shown in Table 1, the enzyme retained approximately 96% of its initial activity after incubation in 10 mM EDTA, suggesting that EDTA did not inhibit the enzymatic activity and NAGaseA is non-metal dependent. Cu 2+ showed a great inhibition effect on the activity of NAGaseA, which was similar to that of NAGases from A. caviae (Cardozo et al., 2017) and C. meiyuanensis (Zhang et al., 2018). Besides, NAGaseA activity was partially inhibited by Fe 3+ and Co 2+ , NAGases from R. miehei and Streptomyces alfalfa shared the same profile as reported (Yang et al., 2014;Lv et al., 2019).

Substrate Specificity of NAGaseA
The substrate specificity of NAGaseA was measured using standard assay conditions. As depicted in Table 2, NAGaseA exhibited the highest specific activity toward pNP-GlcNAc, with a specific activity of 333.33 U/mg. Among (GlcNAc) 2−6 , NAGaseA showed the highest activity toward (GlcNAc) 2 , followed by (GlcNAc) 3 , (GlcNAc) 4 , (GlcNAc) 5 , and (GlcNAc) 6 , which showed that the specific activity toward N-acetyl COSs decreased with increasing degree of polymerization (Ogawa et al., 2006). Besides, little activity (0.0037 U/mg) was detected using colloid chitin as substrate, which was agreed with other reported GH20 NAGases that exhibited little hydrolysis activity toward chitin substrate without the cooperation with other chitinases (Zhou et al., 2017b). Moreover, no activity was observed when chitosan, chitin power, CMC was used as the substrates. These results indicated that NAGaseA possessed the typical NAGase activity with strict substrate specificity.
In addition, the kinetic parameters for NAGaseA were also measured with pNP-GlcNAc as the substrate. The results showed that the V max , K m , k cat , and k cat /K m for NAGaseA were 3333.33 µmol min −1 l −1 , 39.99 µmol l −1 , 4667.07 s −1 , and 116.71 ml µmol −1 s −1 , respectively.

Synergistic Action Between NAGaseA and Chitinases on Chitin Degradation
To investigate the potential application of NAGaseA in GlcNAc production, the synergistic action between NAGaseA and other chitinases on chitin degradation was studied. As illustrated in Figure 5, the released reducing sugar concentrations from the cooperation of NAGaseA with purified chitinase chiA, the crude enzyme from C. meiyuanensis SYBC-H1, and crude enzyme from Chitinibacter sp. GC72 were 0.759, 0.481, and 0.986 g/l, which were 1. 61-, 2. 36-, and 2.69-fold that of the concentration of the two enzymes accumulated, respectively. Among, NAGaseA behaved the best to improve efficiency with the crude enzyme from GC72, which could be attributed to the better synergistic effect with other chitinases from Chitinibacter sp. GC72. Zhou et al. reported a combination of commercial chitinase CtnSg and NAGase rHJ5Nag used for chitin degradation, with an improvement rate of 2.02-fold (Zhou et al., 2017b). Chenyin Lv et al. also investigated the synergistic action between commercial chitinase SgCtn and NAGase SaHEX, which obtained higher production of reducing sugars than the single enzyme for SgCtn (4.3-fold) and SaHEX (8.1-fold; Lv et al., 2019). In our study, NAGaseA can not only combine with purified chitinase but also crude chitinases in the production of GlcNAc from chitin. Moreover, it was worth noting that GlcNAc purity of 96% was obtained and few other N-acetyl COSs were detected in the final reaction mixture. Li et al. (2021) obtained the GlcNAc with a purity of 99.7% using colloidal chitin as the substrate under the co-action of two chitinases after 24 h of incubation, Du et al. (2019) reported the maximum GlcNAc yields of 87% using two enzyme combination during 2.25 h. These results indicate that NAGaseA has great potential in the production of GlcNAc in the multienzyme combination system.

CONCLUSION
In this study, the gene encoding a GH20 family β-Nacetylglucosaminidase NAGaseA from the chitinolytic bacterium Chitinibacter sp. GC72 was cloned and functionally expressed. The domain structure prediction showed that NAGaseA contains GH20 family catalytic domain and exhibited low similarity with reported GH20 NAGases. Analysis from the HPLC revealed that NAGaseA was a multifunctional NAGase exhibited the exo-acting activity and trans-glycosylation activity. Furthermore, NAGaseA also behaved with excellent synergistic performance with other chitinases during the degradation of colloidal chitin, and high purity of GlcNAc was obtained as the final product. These results indicated that NAGaseA has great potential in the bioconversion of chitin waste and behaved as an excellent candidate in GlcNAc production.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.


Characteristics and osteogenic mechanism of glycosylated peptides-calcium chelate

Finding effective practical components to promote bone mineralization from the diet has become an effective method to regulate bone mass. In this study, peptides-calcium chelate derived from Crimson Snapper scales protein hydrolysates (CSPHs), and xylooligosaccharide (XOS)-peptides-calcium chelate prepared by transglutaminase (TGase) pathway, named CSPHs-Ca and XOS-CSPHs-Ca-TG, were used to explore the effects of glycosylation on their structural properties and osteogenic activity in vitro. Results showed that XOS-CSPHs-Ca-TG had better calcium phosphate crystallization inhibition activity with more unified structures than CSPHs-Ca, and could effectively maintain a stable calcium content in the gastrointestinal tract. Meanwhile, the glycosylated peptide-calcium chelate could accelerate the calcium transport efficiency in the Caco-2 cell monolayer, up to 3.54 folds of the control group. Moreover, XOS-CSPHs-Ca-TG exhibited prominent osteogenic effects by promoting the proliferation of MC3T3-E1 cells, increasing the secretion of osteogenic related factors, and accelerating the formation of intracellular mineralized nodules. RT-qPCR results further confirmed that this beneficial effect of XOS-CSPHs-Ca-TG was achieved by activating the Wnt/β-catenin signaling pathway. These results suggested that glycosylation might be a promising method for optimizing structural properties and osteogenic activity of peptide-calcium chelate.

Introduction
Osteoporosis is one of the most common bone diseases and stands sixth among the most frequently-occurring diseases. Its clinical manifestations are reduced bone mass, destruction of bone microstructure, and increased bone brittleness, leading to increased fracture risk . Bone remodeling is achieved by coordinating two cell types with opposite functions, namely osteoclasts and osteoblasts (Zhang et al., 2021). Regularly, bone formation of osteoblasts and resorption of osteoclasts are in a dynamic balance during bone reconstruction. When both values change and tend to resorption, osteoporosis will occur (Jie et al., 2018). Currently, the treatment of osteoporosis is still based on chemical drugs. According to the different action mechanisms, they can be divided into bone resorption inhibitors (bisphosphonates, estrogen, calcitonin), bone formation promoters (fluoride, strontium preparations), and bone mineralization drugs (Vitamin D, calcium preparations). However, various side effects and high costs limit the application of the first two treatments (Tonk et al., 2022). Therefore, finding practical components to promote bone mineralization will be an efficient way to regulate bone mass.
Calcium is the main component of human bones and teeth, 90% of which is absorbed in the small intestine. However, in the weak alkaline environment of the small intestine, calcium ions are inclined to form insoluble calcium salt with acid radical ions in food, leading to the reduction of calcium bioavailability in the human body (Gao et al., 2018a). Therefore, novel calcium supplements emerged as the time required, among which peptides like casein phosphopeptide (CPP)  and phosvitin phosphopeptide (PPP) (Jiang and Mine, 2000) are the prominent representatives. They can combine calcium ions to prevent precipitation, thus increasing soluble calcium concentration in the small intestine (Zhu et al., 2020). Additionally, as the primary calcium carrier, they can interact with the cell membrane and then open the calcium channel to promote calcium absorption in the human body to ameliorate osteoporosis (Lin et al., 2020). Recent studies have demonstrated that peptides extracted from egg yolk soluble protein (Kim et al., 2008), fish collagen (Yamada et al., 2013), and bovine serum  could promote calcium absorption and have osteoprotective activity by inducing the differentiation of pre-osteoblasts. Further studies showed that porcine bone collagen peptide-calcium chelate could up-regulate the expression of osteoblast differentiation markers, such as alkaline phosphatase, type I collagen, and Runx2 . Zhang et al. (2018a) found that the bone mineral density and bone strength of ovariectomized rats fed with Pacific cod peptide-calcium chelate were significantly increased. Similar results were also obtained in the bone metabolism of collagen peptide-calcium chelate in rats with calcium deficiency models by Zhao et al. (2014) and Wang et al. (2018a). These results suggest that peptide-calcium chelate benefits bone metabolism and could be a potential supplement for improving osteoporosis.
Indigestible oligosaccharides are considered the most promising prebiotics for bone health because they can stimulate and activate intestinal flora, promote calcium absorption, improve bone strength and benefit other bone health agents (Arora et al., 2021). Zhang et al. (2021) found that FOS/GOS could significantly improve bone loss in mice. By feeding calcium deficient mice with Konjac oligosaccharide for 8 weeks, Ai et al. (2021) found that the number of trabeculae and bone mechanical strength was heightened. Therefore, indigestible oligosaccharides are also considered a new substance to improve osteoporosis. Moreover, glycosylation is the most common way of protein chemical modification. Based on the advantage of glycosylation modification and the in-depth study of functional peptides, glycosylation has been applied to peptides to improve their physiological activity (Gao et al., 2018b). Zhu et al. (2020) investigated the fabrication of calcium delivery systems by casein phosphopeptides and chitosan oligosaccharides, showing that the delivery vehicles could enhance calcium retention and bone synthesis in mice. However, the knowledge of the mechanism of glycosylated peptide-calcium chelate promoting bone formation is still limited.
Therefore, in the present study, novel glycosylated peptide-calcium chelates derived from Crimson Snapper scales protein hydrolysates (CSPHs) and xylooligosaccharide (XOS) were prepared, and their calcium absorption promoting activity was investigated in the Caco-2 cell model. In addition, the osteogenesis effects and the underlying mechanism of the prepared peptide-calcium chelates were evaluated in the MC3T3-E1 cell model. These results could provide theoretical support for the potential application of peptide-calcium chelates in bone metabolism.

Preparation of crimson snapper scales protein hydrolysates
The scales were dispersed in distilled water (4%, m/V) and autoclaved for 60 min. Before the addition of flavourzyme (E:S = 1:25), the pH value of the mixture was adjusted to 6.30. After being hydrolyzed at 50 • C for 9 h, the hydrolysate was immersed in boiling water for 10 min to inactivate the enzyme. The degree of hydrolysis was 18.21% by formaldehyde titration (Li et al., 2012). Subsequently, the mixture was centrifuged at 10,000 rpm for 20 min, and then the supernatant was collected and freeze-dried. The resulting powder was identified as CSPHs.

Preparation of glycosylated peptide copolymers by TGase
The mass ratio of XOS to CSPHs was 1:0.85, and the total concentration was 30 mg/mL by adding distilled water. Before adding TGase (E:S = 1:10) to the mixture, the pH value of the solution was adjusted to 6.00. Then, the mixture solution was stirred in a water bath for 1.5 h at 40 • C. After that, the complete solution was heated at 95 • C for 10 min to inactivate the enzyme. Then, the solution was cooled to room temperature, put into a dialysis bag, and dialyzed at 20 • C for 24 h (Zhao et al., 2014). Finally, the resulting product was lyophilized. The grafting degree of the freeze-dried powder was 55.62%, calculated by the ortho-phthalaldehyde (OPA) method (Pirestani et al., 2017), and was named XOS-CSPHs-TG.
CSPHs and XOS-CSPHs-TG were respectively dispersed in distilled water to a concentration of 20 mg/mL (pH 7.50). Solid CaCl 2 was added to obtain a samples/CaCl 2 mixture with a mass ratio of 5:1. Then, the mixtures were held at 30 • C for 20 min. To obtain CSPHs and XOS-CSPHs-TG calcium chelates, anhydrous ethanol was added to the mixtures and centrifuged at 10,000 rpm for 20 min. After the precipitates were lyophilized, their calcium-binding rate was determined to be 87.43% and 91.29%, according to the method of Zhang, Lin and Wang (2018b), respectively, which were defined as CSPHs-Ca and XOS-CSPHs-Ca-TG.

Properties and structural characterization of calcium chelating peptides 2.4.1. Inhibition of calcium phosphate crystallization
Each of CSPHs and XOS-CSPHs-TG (20 mg) was dissolved in 2 mL of 0.8 M CaCl 2 solution (pH 7.00) and maintained at 37 • C for 5 min, respectively. Subsequently, 0.008 M NaH 2 PO 4 (200 mL) was added to the solution. The decreasing pH of the solution was recorded every 5 min for 30 min. Distilled water was used as blank control and EDTA as a positive control.

Simulated gastrointestinal digestion experiments
The prepared 1 mg/mL pepsin solution (pH 2.00) and 1 mg/mL pancreatin solution (pH 7.00) were used to simulate the gastric and intestinal fluid. CSPHs-Ca and XOS-CSPHs-Ca-TG were dissolved in distilled water (0.5 mg/mL), respectively, and the pH of the solutions was adjusted to 2.00. After that, 1 mg/mL pepsin solution was added to make the mass ratio of enzyme to chelates 1:50 and was subsequently magnetic stirred at 37 • C for 2 h. Then, the solution was adjusted to pH 7.00 and added with 1 mg/mL trypsin solution to make the mass ratio of enzyme to sample 1:25, followed by maintaining at 37 • C for 2 h with magnetic stirring. During which, 1 mL of solution was taken out every 30 min to calculate the calcium ions retention rate as follows: M (g) was the total calcium content in samples, and M 0 (g) was the calcium content in samples.

Scanning electron microscopy (SEM)
The microstructures of the complexes were observed by SEM (Helios G4 CX, FEI Czech Republic S.R.O., Cernovická Terasa, Czech Republic). The different lyophilized samples were smeared onto an aluminium plate with double-sided adhesive carbon tape and operated at a voltage

Fluorescence spectroscopy analysis
The changes in intrinsic fluorescence were analyzed according to the method of Cai et al. (2015) by a fluorescence spectrometer (Fluoromax-4c-l, Horiba instrument Inc, Piscataway, New Jersey, USA). CSPHs, CSPHs-Ca, XOS-CSPHs-TG, and XOS-CSPHs-Ca-TG were dissolved to 100 μg/mL with deionized water (pH 7.00). The fluorescence spectra were measured at 290-500 nm emission wavelength and 280 nm excitation wavelength, and the slit was 5 nm.
Then, 1 mL of HBSS was added to each well and incubated for 30 min. After detached and resuspending with HBSS, the intracellular calcium concentration [Ca 2+ ] i of the cells was detected by flow cytometry (BD AccuriC6 Plus, BD Bioscience, New Jersey, USA).
The culture medium's volume was 1.5 mL on the basal side and 0.5 mL on the apical side. To evaluate the tight junction permeability of Caco-2 cell monolayer, transepithelial electrical resistance (TEER) values were checked using the Millicell ERS-2 system (Millipore Corporation, Massachusetts, USA). Cell monolayer with a TEER value exceeding 300 Ω cm − 2 was used for the calcium transport experiments. 4 mg/mL CSPHs-Ca and XOS-CSPHs-Ca-TG were added to the apical side, and then the calcium transport capacity across Caco-2 cell monolayers was measured after 2 h. Finally, the calcium content in the basal side was determined by inductively coupled plasma-atomic emission spectroscopy (Lin et al., 2020)    lysates were then sonicated in an ice bath for 5 min and centrifuged at 12,000 rpm for 20 min at 4 • C. The intracellular ALP activity and the total protein content were measured, and the measured intracellular ALP activity was standardized by protein concentration.

Measurements of OCN and Col-I secretions by ELISA
MC3T3-E1 cells were cultured with an osteogenic differentiation medium in the presence of chelates for 7 d or 14 d. After differentiation, the supernatants were harvested to determine OCN and Col-I secretions, respectively.

Mineralization assay
MC3T3-E1 cells were seeded in 6-well plates (Corning Costar, New York, USA) and cultured with an osteogenic differentiation medium with chelates or DKK 1 for 21 d. After cultivation, the cells were fixed in 4% (V/V) paraformaldehyde for 10 min. Cells were then stained with Alizarin Red S (pH 4.2, Sigma Aldrich, St. Louis, Missouri, USA) at room temperature for 30 min. After staining, the wells were washed with distilled water to remove the unbound dye, and staining was visualized using microscopy (Ts2, Nikon Instruments Co., Ltd., Tokyo, Japan). To quantify the bound dye, the cells were destained with 10% (V/V) , and OPG (F) related to Wnt/β-catenin pathway. Different case letters mean a significantly different, p<0.05. "***" represents p<0.001, "**" represents p<0.01, "*" represents p<0.05. cetylpyridinium chloride (Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China) in the dark for 1 h. The solubilized Alizarin Red S concentration was measured at 570 nm using a microplate reader (SpectraMax iD3, Molecular Devices Instrument Co., Ltd., Sunnyvale, USA).

Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) analysis
The MC3T3-E1 cells were treated with an osteogenic differentiation medium with chelates and DKK 1 for 7 d, respectively. Afterward, total RNA was extracted from cells using an RNA preparation pure cell kit (Tiangen Biotechnology Co., Ltd., Beijing, China) and further reversetranscribed into cDNA using a supermax RT reagent kit with a gDNA eraser (Tansgen Biotech Co., Ltd., Beijing, China). RT-qPCR was performed on a real-time PCR system (Bio-Rad Laboratories, California, USA) after adding the primers and SYBR green I to cDNA. The sequences of the primers were shown in supplemental materials Table S1.

Statistical analysis
All experiments were performed in triplicate, and the resulting values were expressed as means ± standard deviations (SD) and analyzed by one-way analysis of variance (ANOVA) using SPSS 22.0 software (SPSS Inc., Chicago, USA).

Properties characterization
The small molecular weight polypeptides produced by enzymatic hydrolysis of proteins have excellent mineral absorption promoting activity and gastrointestinal stability in vitro (Sun et al., 2020). Calcium ion is easy to form insoluble calcium salt with phosphoric acid, phytic acid, and oxalic acid in the intestine, affecting the bioavailability of calcium (Gao et al., 2018b). In the precipitation reaction of Ca 2+ and PO 4 3− , H 2 PO 4− and HPO 4 2− released a lot of H + ; thus, the decreasing pH value can be used to estimate the content of calcium phosphate crystals (Zhu et al., 2020). The pH value of the control group dropped most rapidly (Fig. 1A). With the addition of CSPHs, the pH value decreasing rate was slower, implying that calcium phosphate crystallization was inhibited.
Notably, the effects of XOS and XOS-CSPHs-TG group were superior to CSPHs, which was very close to the effect of EDTA within 10 min of the reaction, indicating that XOS-CSPHs-TG virtually prevented calcium from forming insoluble calcium phosphate. Therefore, it is vital to introduce XOS to form copolymers with CSPHs to improve the calcium-binding ability and further enhance their ability to hinder calcium phosphate crystallization. After gastrointestinal digestion, CSPHs-Ca and XOS-CSPHs-Ca-TG released a tiny amount of calcium ions, indicating that calcium ions enter the intestine in the form of soluble chelates, thereby increasing the overall absorption of calcium (Fig. 1B) (Lin et al., 2020). Similarly, Gao et al. (2018b) found that combining soluble dietary fiber (SDF) with CPP could avoid CPP degradation in the stomach and ensure that CPP bonded to calcium ions so that as many calcium ions could be absorbed in the intestine as possible.

Structural characterization
Aromatic amino acids such as tryptophan, tyrosine, and phenylalanine can generate endogenous fluorescence at specific excitation wavelengths, and the changes in fluorescence intensity can be used to study the conformational changes of peptides and their interactions with small molecules such as metal ions . As shown in Fig. 1C, the fluorescence intensity of CSPHs at 300-320 nm significantly decreased after being conjugated with XOS. Besides, after chelation with Ca 2+ , the fluorescence intensity of the chelate further decreased compared to the corresponding peptides/XOS-peptides, possibly due to the formation of the peptides-Ca complex, which disrupted the conjugated double bond of CSPHs and produced substituent effect. Previous studies have shown that the chelation of metal ions with peptides can lead to endogenous fluorescence quenching, resulting from structural folding and aggregation of peptides during chelation (Cai et al., 2015;Lin et al., 2020). The decreased fluorescence intensity proved that CSPHs folded and aggregated in the glycosylation and chelation reaction with XOS and Ca 2+ . In addition, the emerged fluorescence peak of XOS-CSPHs-Ca-TG at 400-450 nm indicated the formation of glycosylation products .
As shown in Fig. 1D, there were many crystal diffraction peaks in the diffraction patterns of CaCl 2 at certain angles, but no obvious crystal diffraction peaks were observed in the patterns of CSPHs, CSPHs-Ca, XOS-CSPHs-TG, and XOS-CSPHs-Ca-TG. Thus, it could be seen that CSPHs and XOS-CSPHs-TG were not simply mechanically mixed with CaCl 2 . In addition, compared with CSPHs spectra, the peak positions and peak intensities of the spectra of other substances had changed significantly, indicating that new chemical complexes had been formed after glycosylation and chelation reactions (Lin et al., 2019).
As shown in Fig. 1E, CSPHs-Ca was spherical and connected precisely to form a grape cluster with a rough surface, possibly due to the crosslinking interaction effect induced by Ca 2+ Zhu et al., 2020). Moreover, XOS-CSPHs-Ca-TG was also precisely linked together, the shape was more uniform and smaller, and the surfaces became smooth, which indicated that XOS could wrap CSPHs-Ca while XOS-CSPHs-Ca-TG could be well combined through covalent bonds. Therefore, it could be confirmed that the glycosylation reaction has effectively optimized the CSPHs-Ca structure (Wu et al., 2017).
To clarify the structural changes caused by glycosylation and chelation, the FTIR spectra of the samples were compared and analyzed (Fig. 1F). The stretching vibration absorption peak of -OH and N-H is 3427.64 cm − 1 and 2909.74 cm − 1 , respectively, the interval of which is the characteristic peak of XOS (Sudha et al., 2011). The characteristic peaks of saccharides in XOS-CSPHs-TG and XOS-CSPHs-Ca-TG were broader and stronger than those in CSPHs and CSPHs-Ca, which indicated that different glycosylation products were formed. The stretching vibration of C--O caused amide I brand (Miller et al., 2013). After chelating with Ca 2+ , the absorption peak of CSPHs shifted from 1653.68 cm − 1 to 1666.70 cm − 1 ; after glycosylation, the absorption peak also shifted accordingly. Meanwhile, the absorption peak of about 1640 cm − 1 was caused by the stretching vibration of -COO- . Therefore, C--O and -COO-also play an essential role in the covalent binding reaction. Moreover, compared with CSPHs-Ca and CSPHs, glycosylation products showed the shift of absorption peak and the change of intensity at 1200-1000 cm − 1 , indicating that there were more -OH in the copolymer (Geng et al., 2014). Results showed that the TGase could bind XOS to CSPHs. It could be inferred that the amino nitrogen atom, carboxyl oxygen atom, hydroxyl, and carbonyl oxygen atom might be the primary interaction sites of CSPHs and XOS-CSPHs-TG with Ca 2+ (Gao et al., 2018b;Lin et al., 2020;Zhu et al., 2020).

Effects on calcium absorption in Caco-2 cells
Since the chelation reaction is a rapid process, the polypeptides used in this study were premixed with calcium ions to form a chelating solution to act on the cells directly (Hobbs et al., 2001;Lin et al., 2020). As   Fig. 7. Possible mechanism of CSPHs-Ca and XOS-CSPHs-Ca-TG promoting osteoblast proliferation, differentiation, and mineralization (left: inhibited; right: activated). In the presence of CSPHs-Ca and XOS-CSPHs-Ca-TG, Wnt activates the FRZ/LRP5/6 complex and subsequently promotes the expression of Dvl, therefore, leading to the phosphorylation and the degradation of the β-catenin destruction complex, including GSK-3β, APC, and AXIN. The translocation of β-catenin from the cytoplasm to the nucleus functions as a transcriptional co-activator with TCF enhancer LEF and then regulates the target genes, such as Runx2 and OPG. In the presence of the inhibitor DKK1, Wnt signals are suppressed. In the absence of the Wnt ligand, the destruction complex of β-catenin, a tertiary complex formed by AXIN, CK1, GSK3β, and APC, phosphorylates β-catenin, which subsequently undergoes the ubiquitin-proteasomal degradation.
shown in Fig. 2A, CSPHs-Ca and XOS-CSPHs-Ca-TG significantly improved calcium absorption in the Caco-2 cell model (p<0.05). Meanwhile, 8 mg/mL CSPHs-Ca and XOS-CSPHs-Ca-TG enhanced the calcium absorption by 109% and 113%, respectively, compared with the CaCl 2 group (p<0.05), and the effects of which on calcium absorption were equivalent (p>0.05). However, whether CSPHs-Ca and XOS-CSPHs-Ca-TG play the same role in promoting calcium absorption needs further study. Similarly, results were also found on fish scale protein hydrolysate (Lin et al., 2020), soybean hydrolysate (Lv et al., 2008), and tilapia bone collagen hydrolysate (Liao et al., 2020), indicating that calcium-chelating peptide was interacting with the plasma membrane as a calcium carrier to increase calcium influx, thus promoting calcium uptake (Hou et al., 2015;Lin et al., 2020).
The absorption of calcium ions across intestinal epithelium mainly includes two modes: paracellular transport and transcellular transport (Sun et al., 2016). When treated with 4 mg/mL and 8 mg/mL CSPHs-Ca, the calcium transport of CSPHs-Ca was 2.51 and 2.74 folds higher than that of the control group, respectively (Fig. 2B) (p<0.05). There was no significant difference between them (p>0.05), implying that the calcium transport of CSPHs-Ca was saturable. It suggests that the transcellular transport might be involved in CSPHs-Ca to facilitate calcium absorption, which contrasts with the previous reports showing that the paracellular pathway is one of the main pathways of calcium transport across Caco-2 cell monolayer in the presence of peptide-calcium chelates (Sun et al., 2016). With the increase of XOS-CSPHs-Ca-TG concentration, calcium transport increased significantly dose-dependent (p<0.05). While in the presence of 8 mg/mL XOS-CSPHs-Ca-TG, calcium transport was 3.54 folds higher than in the control group (p<0.05). Therefore, the main pathway of the XOS-CSPHs-Ca-TG encouraging calcium absorption in cell monolayer may differ from that of CSPHs-Ca, and it should be the paracellular cell pathway (Liao et al., 2020). Notably, after glycosylation, the calcium transport capacity of XOS-CSPHs-Ca-TG was remarkably higher than that of XOS and CSPHs (p<0.05), indicating that glycosylation is an effective means to enhance the calcium absorption of peptide-calcium chelate.

Effects on the proliferation of MC3T3-E1 cells
The proliferation process of pre-osteoblasts is the premise of forming new bone in bone tissue, which is also the critical process affecting bone anabolism in the body (Kenichi et al., 1998). The MC3T3-E1 cells model was used to assess the osteogenic activity of peptide-calcium chelates in vitro. As shown in Fig. 3, the cell viability of MC3T3-E1 cells was remarkably up-regulated to about 120%-155% after being cultured for 24-72 h in CSPHs-Ca and XOS-CSPHs-Ca-TG. As shown in supplemental materials Table S2, when treated with CSPHs-Ca and XOS-CSPHs-Ca-TG, the G0/G1 phase ratio decreased immensely, while the ratio of the S phase increased (p<0.05). Meanwhile, compared with CSPHs-Ca, XOS-CSPHs-Ca-TG had a more notable cell cycle stimulation effect (p<0.05). Altogether, the proliferation of MC3T3-E1 cells stimulated by CSPHs-Ca and XOS-CSPHs-Ca-TG was related to the regulation of the cell cycle, which accelerated the transformation from the G0/G1 phase to the S phase, advanced DNA synthesis, and thus boosted cell proliferation .

Effects on ALP activity in MC3T3-E1 cells
ALP is an external enzyme of osteoblasts, and its expression activity is a remarkable feature of osteoblast differentiation (Jie et al., 2018). As shown in Fig. 4A, ALP activity of MC3T3-E1 cells treated with CSPHs-Ca and XOS-CSPHs-Ca-TG was significantly higher than that of the control group (p<0.05), and the ALP activity were highest in XOS-CSPHs-Ca-TG groups. That might be because XOS-CSPHs-Ca-TG contains more hydroxyl groups after glycosylation, which could better interact with MC3T3-E1 cell surface receptors, the "external-internal signal" on MC3T3-E1 cells (Jie et al., 2018). In other words, more hydroxyl groups in XOS-CSPHs-Ca-TG enhance cell interaction and trigger signal conversion, thereby stimulating cell differentiation through receptor-mediated mechanisms (Jie et al., 2018;Liu et al., 2013). Interestingly, it takes almost 6 d for MC3T3-E1 cells to differentiate into mature osteoblasts. The ALP activity in all groups on day 14 was lower than on day 7, which might be because ALP is an early phenotypic marker during osteoblast differentiation (Wang et al., 2012). As the differentiation time is prolonged, ALP might catalyze the hydrolysis of bone phosphate monoester to provide inorganic phosphates, which bind to calcium to regulate bone metabolism and mineralization (Jie et al., 2018).

Effects on OCN and Col-I secretion in MC3T3-E1 cells
OCN and Col-I are biochemical markers of bone metabolism . OCN is necessary for osteoblasts to regulate the process of mineralization, and Col-I is the major protein in the bone matrix, synthesized by osteoblasts and involved in differentiation (Willett et al., 2014). After 7 d of differentiation, the secretion of OCN and Col-I in MC3T3-E1 cells treated with CSPHs-Ca and XOS-CSPHs-Ca-TG was significantly higher than that of the control group (p<0.05) (Fig. 4B-C). After 14 d of differentiation, the content of OCN and Col-I in all treatment groups continued to increase concentration-dependent (p<0.05). Moreover, the cells treated with XOS-CSPHs-Ca-TG had higher secretion (p<0.05). It suggests that CSPHs-Ca and XOS-CSPHs-Ca-TG could enhance OCN and Col-I secretion in osteoblasts and further promote differentiation of osteoblasts in early and mature stages .

Effects on mRNA expression of Wnt/β-catenin pathway-related factors
The canonical Wnt signaling pathway is essential in regulating bone homeostasis, which controls the proliferation and differentiation of osteoblastic precursors and maintains mature osteoblasts (Wu et al., 2019). It was known that Wnt3a could activate the canonical Wnt signaling pathway (Ikehata et al., 2017). Wnt3a promotes Dishevelled (DVL) expression by activating the Frizzled/LRP5/6 complex, which leads to the phosphorylation and the degradation of the β-catenin destruction complex (including glycogen synthase kinase 3β (GSK-3β), adenomatous polyposis coli (APC), and Axis inhibition protein (AXIN)) (Hwang et al., 2009). Subsequently, the translocation of β-catenin from the cytoplasm to the nucleus functions as a transcriptional co-activator with T cell factor enhancer lymphoid enhancer factor (TCF/LEF) and then regulates the target genes, such as Runt-related transcription factor 2 (Runx2) and osteoprotegerin (OPG), both key transcription factors associated with the expression with osteoblast differentiation (Hwang et al., 2009;Wu et al., 2019). As shown in Fig. 5, compared to the control group, the mRNA expression of Wnt3a, LRP5, β-catenin, Runx2, and OPG was significantly increased after being treated with CSPHs-Ca and XOS-CSPHs-Ca-TG (p<0.05), while the expressions level of GSK-3β was gradually inhibited dose-dependent. Meanwhile, the XOS-CSPHs-Ca-TG has a more significant effect (p<0.05). That evidence strongly suggests that glycosylated peptide-calcium chelate plays a positive role in osteogenesis.
To confirm that the canonical Wnt signaling pathway is a stimulator and regulator of osteogenesis, the cells were incubated with DKK 1 (binds to LRP5/6 co-receptor and inhibits canonical Wnt/β-catenin signaling) before being treated with CSPHs-Ca and XOS-CSPHs-Ca-TG. As shown in Fig. 6, pretreatment with DKK 1 reduced ALP activity (Fig. 6A) and mRNA expression level of osteogenic transcription factor stimulated by CSPHs-Ca and XOS-CSPHs-Ca-TG to the control level ( Fig. 6B-E) (p<0.05). To sum up, the Wnt/β-catenin signaling pathway regulates the differentiation of MC3T3-E1 cells stimulated by CSPHs-Ca and XOS-CSPHs-Ca-TG (Fig. 7).

Effects on mineralized nodules of MC3T3-E1 cells
Mineralization is the last step of osteoblast differentiation, directly reflecting osteogenesis's degree (Jie et al., 2018). The calcium ions in the mineralized nodule are a dark red color after complexation with alizarin red S, and its absorbance is directly proportional to the amount of calcium deposition . Compared to the control group, both treatments in 400 μg/mL could produce a small number of mineralized nodules, while XOS-CSPHs-TG had a more significant effect (p<0.05) (Fig. 8A). After adding 400 ng/mL DKK 1, the number of mineralized nodules in each treatment group decreased significantly, which was still slightly higher than that in the control group (p<0.05) ( Fig. 8A-B). When 4 mM CaCl 2 was premixed with 400 μg/mL CSPHs and XOS-CSPHs-TG to form chelates, the number of mineralized nodules increased significantly, and the XOS-CSPHs-Ca-TG had a more significant effect (p < 0.05) (Fig. 8B). It could be explained by the fact that the calcium-binding capacity of the XOS-CSPHs-Ca-TG was better than CSPHs-Ca (data not shown), which could provide more calcium ions and higher-density nucleation sites for the formation of minerals, leading to reduced interfacial energy generated by minerals, in turn, promote the mineralization process (Jie et al., 2018;Wu et al., 2020). Thus, CSPHs-Ca and XOS-CSPHs-Ca-TG regulate the proliferation, differentiation, and mineralization of MC3T3-E1 cells by activating the Wnt/β-catenin signaling pathway.

Conclusions
In this study, the structural properties and osteogenic activity of glycosylated peptide-calcium chelate were firstly investigated. Compared with CSPHs-Ca, the XOS-CSPHs-Ca-TG had better calcium phosphate crystallization inhibition activity with more unified structures. The formation of XOS-CSPHs-Ca-TG avoided digestion in the stomach and dramatically improved the calcium transport efficiency in the Caco-2 cell monolayer. Moreover, XOS-CSPHs-Ca-TG showed excellent osteogenic activity, which could effectively promote the proliferation, differentiation, and mineralization of MC3T3-E1 cells, and this beneficial effect of XOS-CSPHs-Ca-TG was achieved by activating the Wnt/β-catenin signaling pathway. Thus, XOS-CSPHs-Ca-TG plays a pivotal role in improving/preventing osteoporosis and can be used as a functional food ingredient to prevent bone metabolism-related diseases.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Preliminary Expression Analysis of the OSCA Gene Family in Maize and Their Involvement in Temperature Stress

Hyperosmolality-gated calcium-permeable channels (OSCA) are characterized as an osmosensor in plants; they are able to recognize and respond to exogenous and endogenous osmotic changes, and play a vital role in plant growth and adaptability to environmental stress. To explore the potential biological functions of OSCAs in maize, we performed a bioinformatics and expression analysis of the ZmOSCA gene family. Using bioinformatics methods, we identified twelve OSCA genes from the genome database of maize. According to their sequence composition and phylogenetic relationship, the maize OSCA family was classified into four groups (Ⅰ, Ⅱ, Ⅲ, and Ⅳ). Multiple sequence alignment analysis revealed a conserved DUF221 domain in these members. We modeled the calcium binding sites of four OSCA families using the autodocking technique. The expression profiles of ZmOSCA genes were analyzed in different tissues and under diverse abiotic stresses such as drought, salt, high temperature, and chilling using quantitative real-time PCR (qRT-PCR). We found that the expression of twelve ZmOSCA genes is variant in different tissues of maize. Furthermore, abiotic stresses such as drought, salt, high temperature, and chilling differentially induced the expression of twelve ZmOSCA genes. We chose ZmOSCA2.2 and ZmOSCA2.3, which responded most strongly to temperature stress, for prediction of protein interactions. We modeled the calcium binding sites of four OSCA families using autodocking tools, obtaining a number of new results. These results are helpful in understanding the function of the plant OSCA gene family for study of the molecular mechanism of plant osmotic stress and response, as well as exploration of the interaction between osmotic stress, high-temperature stress, and low-temperature stress signal transduction mechanisms. As such, they can provide a theoretical basis for crop breeding.

Introduction
As a major food crop, maize (Zea mays L.) plays a vital role in solving world hunger problems. However, the production process of maize is highly dependent on suitable environmental factors [1]. With the reduction in the availability and quality of arable land and water resources, as well as frequent extreme weather [2], many different types of abiotic stresses have been highlighted, such as salinity, drought, heat, and chilling [3]. These major abiotic environmental stresses seriously affect crop development and constrain agronomic yields worldwide. Supposedly, abiotic stresses may be responsible for a yield reduction of over 50% in major crop plants globally [4]. Studies have shown that the decline in maize yield is very significant under conditions of undesirable stress. In the last 30 years, maize yield losses due to heat stress were 1.13% per decade in China [5]. Based on reliable data, future high temperatures are predicted to increase the decline in corn production from 7% to 31% [6]. High temperatures are currently a major constraint on corn yields in Northeast China [7]. Cold stress limits food production, and is detrimental to normal plant growth and development [8]. Low temperatures disrupt photosynthetic metabolism, and may produce stress products such as reactive oxygen species, leading to a reduction in nutrients as well as inducing harmful structural changes in cells [9]. Maize growth in the field is influenced by various environmental factors, and it is particularly relevant to elucidate the signaling patterns associated with resistance to stress.
Temperature, salt, and drought stresses are major environmental factors that affect growth and development of plants in world, restrict plant productivity, and threaten human food security. How plants sense these stress signals and adapt to adverse environments are fundamental biological questions [10]. Under osmotic stress, an increase in intracellular calcium levels is achieved through transport pathways such as calcium channels [11]. Plants are subject to adversity stress, which triggers calcium oscillations in plants [12]; at the physiological, cellular, and molecular levels, this takes the form of inducing the expression of many stress-related genes, regulating plant tolerance to stress, and generating specific calcium signals [13]. It has been reported that calcium-associated proteins are required for several environmental stress responses [14]. Ion channels are proteins located in the cell membrane that aid in ion transport and play an important role in maintaining cellular homeostasis [15]. Hyperosmolality-gated calcium-permeable channels (OSCA), as one of the more recently discovered calcium channels, play an essential role in calcium signaling. Following the discovery and identification of OSCA1.1 in Arabidopsis thaliana [16], the OSCA gene family has been increasingly valued by scientists worldwide. Yuan et al. [16] established that OSCA1 protein, localized in the plant cell membrane, is expressed in both leaves and roots of Arabidopsis. Currently, ZmOSCA is associated with osmolarity-related stresses [17] such as drought and ABA, which is consistent with studies on other species. Over time, researchers have discovered that OSCA is widely distributed in eukaryotic cells, whether plants or animals [18]. It has been discovered that OSCA carries a major conserved protein, DUF221, which contains six predicted TM helices [19]. The TM helices in the structures of such proteins may have disappeared during evolution, as OsOSCA2.2 lacks three TM regions [20]. The same study found that OSCA proteins may have effects on other traits. TaOSCA1.4, which belongs to the same family as AtOSCA1. 8 and OsOSCA1.4 in the evolutionary tree analysis, is related to the grain number per ear [21]. The number of grains on the ear is one of the most important traits for maize yield. In addition, osmotic pressure affects the water balance in the plant, which in turn determines the crop yield. OSCA is a confirmed gene family associated with osmolality, and we found that it may have a key role in coping with the effects of temperature as well. This is an important means of improving the knowledge of abiotic stress resistance in maize. More importantly, determining the stress tolerance mechanism of maize can provide a theoretical basis for breeding drought-resistant varieties and improving cultivation techniques.
In the present study, we performed a bioinformatics analysis of the entire maize genome and identified twelve OSCA genes. These twelve maize OSCAs were grouped based on their phylogenetic relationships and their expression profiles in various organs and under different abiotic stresses. We found that OSCAs play a role in osmolality, and may be important in temperature stress and related signaling pathways as well. Furthermore, we predicted the calcium binding sites in proteins and carried out a protein interaction analysis, verifying that our conjecture is possible. These results can be used for functional validation studies of ZmOSCA and increase our understanding of the roles of plant OSCAs.

Phylogenetic Relationship and Gene Structure of Maize OSCAs
We used the CDS (coding sequence of amino acids in protein) of 38 OSCAs from rice, Arabidopsis, and maize to construct a phylogenetic tree ( Figure 1) to determine the evolutionary relationships of OSCA in maize, and collated the information related to the ZmOSCA gene in the maize genome database (http://ensembl.gramene.org/, revised 18 January 2022) ( Table 1). We found that members of the OSCA family were separated into four distinct clades, designated I, II, III, and IV ( Figure 2). Most OSCA family members in Arabidopsis, rice, and maize were mainly distributed in groups I and II as compared with groups III and IV. By further analyzing the results, we found that the OSCA genes in maize and rice were more closely related. We grouped all the OSCAs genes as shown in Table 2. Figure 1. Phylogenetic analysis of OSCA proteins. Arabidopsis, rice, and maize are denoted by circle, diamond, and triangle respectively, and the tree is divided into four groups.   Then, we used Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/index.php, revised 30 January 2022) to generate the intron and exon structure information maps of each gene in the ZmOSCA family ( Figure 2). It was recognized that the gene structures of OSCA genes belonging to the same subfamily were often very similar, with the number and size of introns and exons being nearly identical. Except for ZmOSCA3.1, which contained six exons and for which the fourth exon was significantly longer, all members of the ZmOSCA I subfamily contained eleven exons and ten introns; however, the lengths of exons 1, 2, 5, and 6 of ZmOSCA3.1 are noteworthy. The lengths of exons 1, 2, 5, and 6 of ZmOSCA3.1 are the same as the lengths of the exons at both ends of the other members. All OSCA II subfamily members contain ten exons and nine introns. ZmOSCA4.1 is more distantly related to the other members, and may be more primitive and conserved.

Analysis of Structural Domains of the ZmOSCA Family
DUF221 is a characterized structural domain of the OSCA gene family [16]. Multiple sequence alignments of OSCAs were performed using DNAMAN software (http: //www.lynnon.com/dnaman.html, revised 20 February 2022), and the transmembrane (TM) region of the DUF221 conserved region was predicted using TMHMM. We found that different OSCA members contain different TMs; most of the OSCA have six TMs with two exceptions. ZmOSCA4.1 had fewer TM4, and ZmOSCA1.5 had more TM5 disappear than their orthologues, which suggested that a deletion event occurred in the genomes of ZmOSCA4.1 and ZmOSCA1.5 ( Figure 3). TMs from the same family are similar, while there are large differences between families.

Predicted Secondary Structure Analysis of ZmOSCA Gene Family Proteins
The secondary structure of each member of the ZmOSCA protein family was predicted by SOPMA based on the Protein Data Bank, and the results were counted. The results showed that the twelve OSCA protein family members of maize mainly contained four structures, Alpha helix (Hh), Extended strand (Ee), Beta turn (Tt), and Random coil (Cc), among which Alpha helix (Hh) accounted for the largest number and proportion of the secondary structures ( Figure 4: Table 3). The protein secondary structures of ZmOSCA1.1, ZmOSCA1.2, and ZmOSCA1.3 are identical to each other. ZmOSCA2.1, ZmOSCA2.2, and ZmOSCA2.3 are very close to each other as well. In addition, we discovered a large difference in the secondary structure of proteins from different families.  . Predicted secondary structure of maize OSCA proteins. The blue, red, green, and yellow stripes represent the α helices, the β bridges, the β turns, and the random coils, respectively.

Characterization of the ZmOSCA Gene Family for Tissue-Based Expression
To unveil the potential function of ZmOSCAs in maize, the expression profiles of ZmOSCA genes in various tissues and organs were first determined using RT-PCR (real-time reverse transcription-PCR), and are represented in greyscale to facilitate visualization. The twelve ZmOSCA genes showed tissue-specific expression patterns. ZmOSCA genes were highly expressed in all tissues tested with two exceptions ( Figure 5), which is indicative of a universal role of OSCAs in osmotic-sensing processes throughout the plant. ZmOSCA2.3 and ZmOSCA2.5 were detected only in the leaf, indicative of a specific function therein. ZmOSCA1.3, ZmOSCA1.4, ZmOSCA1.5, ZmOSCA2.3, and ZmOSCA2.4 showed medium transcript abundances in all tissues tested. ZmOSCA1.2, ZmOSCA2.1, and ZmOSCA3.1 had high transcript abundance in the root and relatively high transcript abundance in the stem, suggesting that they are likely related to root uptake. These results indicate that ZmOSCAs are involved in various physiological and developmental processes in maize. Figure 5. Schematic representation of the organ-specific expression of the ZmOSCA genes. The colors white, 25% grey, 35% grey, 50% grey, 75% grey, and black represent the multiple ranges of ZmOSCA mRNA expression levels, which were <0.01, 0.01-0.1, 0.1-1, 1.0-10, 10-50, and >50, respectively, compared with 0.002 GAPDH.

Expression Profiles of ZmOSCAs under Osmotic-Related Abiotic Stresses
To determine whether the expression of ZmOSCAs is responsive to osmotic-related abiotic stress, qRT-PCR analysis of the ZmOSCAs at the 10-day stage in maize was performed under PEG 6000 (10%) and NaCl (250 mM). We found that twelve ZmOSCA genes were down-or upregulated in the stress conditions examined as compared with the control (Figures 6 and 7). Majority of the ZmOSCAs were substantially altered within 6 h. ZmOSCA1.1 was upregulated by 10% PEG and salt stress treatment, and ZmOSCA1.5 was specifically downregulated. ZmOSCA1.3 and ZmOSCA2.1 were downregulated at 1 h and then slowly recovered. Among the genes expressed after treatment, ZmOSCA1.1 was the most altered. These results indicate that ZmOSCAs might be involved in osmotic-related signaling pathways.

Expression Profile of ZmOSCAs under Environment-Related Abiotic Stresses
To further investigate whether ZmOSCAs affect other environmentally relevant abiotic stresses, we used qRT-PCR analysis at the 10-day stage in maize, performed at 4 • C and 40 • C. The test results showed that twelve ZmOSCA genes were changed in the conditions examined as compared with the control (Figures 8 and 9).  When the plants were under 40 • C stress, we found that the expression of OSCA genes could be divided into two groups. The expression trends of six genes, ZmOSCA1.4, ZmOSCA2.1, ZmOSCA2.2, ZmOSCA2.5, ZmOSCA3.1, and ZmOSCA4.1, were relatively similar; all of them showed a significant increase in gene expression level after 1 h of high-temperature stress, reached a peak, and then were rapidly downregulated, followed by a slow increase. The expression trend of ZmOSCA2.1 was similar to that of ZmOSCA2.3, except that the expression level continued to be downregulated until 24 h of treatment, and did not increase. The expression trend of ZmOSCA1.2 was similar, with the difference that expression was slightly downregulated at 1 h of high-temperature stress and then increased continuously.
The expression patterns of ZmOSCA1.3, ZmOSCA1.5, and ZmOSCA2.4 were opposite to the other members of the maize OSCA family. The expression of ZmOSCA1.3 continued to be downregulated after high-temperature stress in maize, and started to show an upregulation trend after reaching a minimum value at 6-12 h. By 24 h, it exceeded its level in the absence of stress. ZmOSCA1.5 expression dropped to a very low level within 1 h of heat stress, then increased slowly during the next 23 h. The initial expression level was restored after 24 h of stress. The expression image of ZmOSCA2.4 showed that after the rapid upregulation of expression level induced by high temperature, the transcript level of this gene continued to be stable at the level above and below three times the level of the initial expression.
From the data obtained from seedlings under low-temperature stress at 4 • C, we know that for the vast majority of ZmOSCA genes (ZmOSCA1.2, ZmOSCA1.3, ZmOSCA1.5, ZmOSCA2.4, ZmOSCA3.1, and ZmOSCA4.1), expression decreases first, then rises to a relatively large value, then gradually decreases again. ZmOSCA1.4 is very similar to this trend, except that it rises to a value and then does not fall. Similarly, ZmOSCA2.1 is not obvious at the beginning of the decline. Their function in plant resistance to low temper-atures is similar as well. More special are ZmOSCA1.1, ZmOSCA2.2, and ZmOSCA2.3. ZmOSCA1.1 showed a cliff-like decrease and only slowly recovered after 12 h. The expressions of ZmOSCA2.2 and ZmOSCA2.3, in general, increased steadily. The expression level of ZmOSCA3.1 at 6 h reached 1.5-fold that in the absence of stress, and we speculate that the transcription of this gene may be regulated by the feedback mechanism of low-temperature stress and involved in a specific function in the low-temperature stress response.

Prediction of Binding Sites for Ca 2+ in OSCA Proteins
As the crystal structure of ZmOSCAs has not been reported, we used the OSCA1.1 of Arabidopsis. We acquired the projected structure of this protein by SWISS-MODEL Repository (OSCA1.1, ID: 6JPF) and modeled the likely binding mode of calcium ions to the OSCA proteins using molecular docking. There are different potential binding sites between calcium ions and OSCAs through polar contacts, as shown in Figure 10. According to the results of the images, we conclude that OSCA is often combined with ASN and ILE. Such a phenomenon may be associated with the charge distribution of protein amino acid residues. Perhaps a deeper study of OSCA, a calcium-permeable protein, could be performed through calcium-binding sites.

ZmOSCA2.2 and ZmOSCA2.3 Protein Prediction Analysis Results
Based on the OSCA genes affected by temperature stress, we selected ZmOSCA2.2 and ZmOSCA2.3, for which the expression was significantly altered at both 40 • C and 4 • C. These were further analyzed in order to predict protein interactions (Figure 11; Tables 4 and 5). From the predictive analysis, we know that the PPI enrichment p-value is 9.36 × 10 -5 for ZmOSCA2.2, while for ZmOSCA2.3 PPI it is 6.58 × 10 -8 . The mean clustering coefficients of ZmOSCA2.2 and ZmOSCA2.3 are 0.793 and 0.855, respectively. Looking at the predicted results, our analysis perceives that both ZmOSCA2.2 and ZmOSCA2.3 have interactions with GRMZM2G029546, GRMZM2G045854, GRMZM2G033926, GRMZM2G175423, GRMZM2G702253, GRMZM2G060296, and GRMZM2G319454. Analysis of its protein annotation and family revealed that GRMZM2G029546 is a Dnaj heat shock n-terminal domain-containing and Electron transporter/heat shock protein binding protein with important roles in plant stress responses and signaling. GRMZM2G045854 is a Gbf-interacting protein, which has a DUF1296 domain, which can enhance the transcriptional activity of plant growth-related transcription factors [22]. GRMZM2G033926 belongs to the 1-acyl-sn-glycerol-3-phosphate acyltransferases, which can catalyze the acylation of lysophosphatidic acid and are responsible for the de novo production of phosphatidic acid [23]. GRMZM2G175423 belongs to the class of Sorbitol dehydrogenases. For enzymes, including many proteins that are irreplaceable in biological reactions, the differences they show with changes in temperature are extremely significant. OSCA has several interactions with a wide range of enzymes in maize.
Moreover, OSCA2.2 interacts closely with the NAD(P)-dependent dehydrogenase, short-chain alcohol dehydrogenase family and allantoin: proton symporter activity. As such, we speculate that OSCA2.2 is related to energy metabolism. In the prediction results of OSCA2.3, two protein families, pectinesterase inhibitor activity and ethylene-activated signaling pathway, were prominent. We speculate that OSCA2.3 is involved in the hormone pathway in plants and may play a specific role. Furthermore, OSCA2.3 was found to be associated with ABC transporter protein. In Gram-Positive Pathogens, the ABC protein is governed by the treatment temperature [24].  Signal recognition particle receptor subunit alpha; Signal recognition particle protein subunit 9 625 COG0552 Signal recognition particle GTPase   Abc transporter c family mrp4 precursor; ABC transporter that may affect phytic acid transport and compartmentalization.

COG1132
The ABC-type multidrug transport system, ATPase and permease component In addition, botanists have shown that the protein expression of ABC transporter protein changes in plant leaves with the impact of temperature [25]. These findings offer additional proof that OSCA is involved in many physiological activities in plants.
Finally, OSCA might be temperature-related in a way that we do not yet know. Our work demonstrates the dependence between OSCA and temperature, and provides evidence to further investigate the temperature mechanism of plants.

Discussion
Ion channels are essential for cell survival, and cells use them for nutrient uptake, sensing environmental stimuli, and signaling [26]. Many different stimulus-sensing proteins exist in plants to sense different stress signals; the functional proteins of the early signaling phase of plant stress signal transduction are unknown. However, it has been demonstrated that low temperature, drought, and high salt stress can induce transient calcium inward flow in the cytoplasm. Thus, it can be inferred that the ion channels causing inward calcium flow are likely to act as a class of receptors for stress signals [27]. The Sodium Proton Antiporter (NHX) temperature of Arabidopsis has previously been found to affect the translation of its gene family, while predictive analysis found that SbNHX is closely linked to the calcineurin B-like proteins (CBL)-interacting protein kinases (CIPK) pathway [28]. This further suggests that calcium proteins may be temperature-dependent. Calcium transporter protein ANN1 was found to be involved in plant cold-induced responses in Arabidopsis [29], suggesting that calcium transporter proteins can be directly involved in plant stress responses due to temperature changes. We found differences in the expression levels of the twelve ZmOSCA genes under high and low temperature conditions, something that has never previously been suggested.
In addition to the above, there are many transcription factors associated with osmoregulation in plants, such as DREB, MYC/MYB, and WRKY [30]. As osmoregulation-related transcription factors, they have been found to play a role in osmosis and to be inextricably linked to other metabolic pathways in plants. DREB2A regulates plant heat stress response by regulating the cascade response of the heat stress transcription factor HsfA [31]. MYB/MYC, the largest family of transcription factors in plants [32], can alter the regulation of stomatal movement and is involved in physiological regulation related to plant sensibility [33]. WRKY has been shown to have an important relationship with the response to high-temperature stress. WRKY factors can bind to the W-box cis-acting elements of target gene promoters, thereby regulating the expression of multiple target genes and participating in multiple signaling pathways in plants [34]. While OSCAs are found to be associated with osmosis, they should not act only in osmoregulation, as with the factors mentioned above. Three OsOSCAs are associated with circadian regulation, and are related to transpiration regulation [20]. The SlOSCA gene regulates light responses and contains elements involved in the light pathway [35]. Based on these results, the role of OSCAs stretches far beyond what has been found to date. OSCAs encode genes that may play a decisive role in other processes of plant growth and development, and these questions require further investigation. In the case of heat stress, changes in other factors provide us with a new viewpoint about OSCAs; based on existing knowledge of OSCAs, we assume that they have a high probability of reacting to thermal stress.
The evolutionary tree we constructed based on the proteins tells us that OSCAs can be classified into four categories, which is consistent with the evolutionary analysis of Arabidopsis, rice, poplar, and grapevine [16]. The OSCA gene family of the typical monocot plant rice and the dicotyledonous plant Arabidopsis is present in each category, showing that the OSCA gene family has the potential to diversify before plant differentiation. We found that the second subfamily of ZmOSCA was expressed in a similar trend in cold stress, which is similar to the results of the promoter analysis in [17]. Thus, we believe that OSCA subfamily II may be a group with less alteration in the biological evolution of the OSCA gene family in terms of their protein reaction to temperature stress. This may play a decisive role in the investigation of species genesis.
Through predictive analysis of ZmOSCA protein interactions, we found that ZmOSCA2.3 is closely associated with catabolic processes such as amino acids, cellular nitrogen compounds, and organic cyclic compounds, suggesting that ZmOSCA2.3 is involved in many metabolic activities. This echoes the OSCA2.3 related study of Thor. et al. [36]. These data strengthen the credibility of our protein interactions prediction analysis. At the same time, we discovered that ZmOSCA involves enzymatic reactions in a variety of organisms. The temperature-enzyme relationship gives us more confidence that ZmOSCA is a protein involved in temperature regulation. In fact, several in vivo transporters have a co-operative relationship with ZmOSCA2.2, and many of these are transmembraneassociated protein transporters. The effect of temperature on the association between plant plasma membrane transport and protein transporters has been well documented in plant physiology. In addition, the properties of protein denaturation at high temperatures allows us to be more certain that ZmOSCA is highly temperature modulation-related.
At present, we have not explored the specific targets of the OSCA signaling pathway under temperature stress via other experiments, and this is the direction we are considering following in continuing our in-depth study in the future. In our protein interactions prediction, we found that OSCA is closely linked to other metabolic pathways, and in the future we will consider validating the role of OSCA within other metabolic pathways in order to explore and discuss more functions that OSCA may have. This protein could be interchangeable with yield-related influences in experiments to help solve the problem of world hunger.

Gene Screening and Identification Analysis
Identified Arabidopsis OSCA gene family gene IDs were downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, revised 17 January 2022), and candidate genes were identified by tBlast N searches in their maize genome library. The ZmOSCA gene family members were identified using the Pfam and SMART databases and validated by the DUF221 structural domain. When multiple transcripts existed at the same gene locus, the deduced amino acid sequence of the longest transcript was selected for comparison. Prediction of genomic exons and introns was carried out using Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/, revised 26 January 2022).

Construction of Evolutionary Tree and Analysis of Gene Structure
The full-length proteins were analyzed using MEGA5.0 software, and phylogenetic trees were constructed by the Neighbor Joining (NJ) method [37]. A total of 5000 bootstrap replicates were performed in each analysis to obtain confidential support. Gene structures of OSCAs were identified using the Gene Structure Display Server. Multiple sequence alignments of OSCAs were performed using DNAMAN, and the transmembrane region of the DUF221 conserved region was predicted using TMHMM.

Analysis of Protein Secondary Structure Prediction in Maize OSCA Family
Based on the principle of homology prediction, the protein secondary structure prediction website SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/ npsa_sopma.html, revised 2 March 2022) was used to predict the secondary structure of OSCA in maize based on the relevant protein database. The secondary structure of each member of the OSCA protein family was predicted based on the relevant protein database, and the results were checked statistically. The TM domains in OSCAs were annotated according to TMHMM Server v. 2.0 predictions (http://www.cbs.dtu.dk/services/ TMHMM/, revised 8 March 2022).

Predictive Analysis of Protein Interactions
Using the STRING website (https://string-db.org/, revised 18 March 2022), the minimum required interaction score was manually set to 0.4 and the first interacting protein shell parameter was set to 30 to construct the protein interaction prediction analysis data, then edited with Cytoscape software and checked with protein information from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, revised 18 March 2022).

Prediction of Recognition Sites for Calcium Ions
The OSCA protein structure was located and downloaded from the PDB database (https://www.rcsb.org/, accessed on 22 March 2022); using the SWISS-MODEL website (https://swissmodel.expasy.org/, accessed 24 March 2022), the remaining several OSCA structures that were not yet measured were constructed homologously using AtOSCA1.1 (ID: 6JPF) as a template [38]. The proteins were then processed using Auto Dock 4.0 [39] for mapping and analysis based on the system-defined highly probable docking sites.

Plant Materials and Growth Conditions
Maize plants (B73) were planted in a growth chamber at Jilin University (Changchun, China). Plants were surface sterilized in a 10% (v/v) H 2 O 2 solution for 20 min and germinated on sand for 3 days in a growth chamber at 4 • C with a 10 h/14 h light/dark cycle and 70-80% relative humidity. The seeds were transferred into improved Hoagland solution at 28/25 • C (day/night) with a photoperiod of 16/8 h (day/night) and relative humidity of 50-70%; the nutrient solution was replaced regularly with a new one every day. When10 days old, maize seedlings were placed in a nutrient solution containing 10% PEG for drought stress treatment; seedlings were treated with a low temperature of 4 • C and a high temperature of 40 • C for 24 h in the light incubator and taken at 0 h, 1 h, 6 h, 12 h, and 24 h after treatment. Maize seedlings were treated with 250 mmol/L NaCl solution for salt stress, and the seedlings were taken at 0 h, 1 h, and 6 h, after treatment. The nutrient solution was renewed daily.

RNA Extraction and Quantitative qRT-PCR Analysis
Extraction of maize RNA was carried out by the TRIzol method. Reverse transcription kits from Invitrogen were then used to synthesize the first strand cDNA. cDNA template was diluted and used and real-time fluorescence quantitative PCR was performed using the SYBR GREEN method as provided by the company's QRT kit. Finally, the relative amount of gene expression was calculated using the 2 −∆∆Ct method [40]. For detailed primer sequences, see Table S1

Conclusions
We found evidence for the involvement of ZmOSCAs in temperature correlation, along with an exhaustive analysis of the ZmOSCA family. We found that the OSCA gene family of maize is characterized by tissue-specific expression, which is highly consistent with studies of OSCA in rice [20]. This is the first discovery in maize, and represents a further exploration of the molecular mechanisms underlying the interactions of ZmOSCA under adversity stresses. Further investigation of the regulatory mechanism of ZmOSCA2.2 and ZmOSCA2.3 in the cold/heat stress response can contribute to the engineering of cold/heat-tolerant crops via element-specific genome editing.